This invention relates generally to the field of methods of encapsulation of physiologically active substances. More specifically, the invention relates to emulsification processes for preparing multivesicular liposome formulations, with sustained release characteristics and the formulations produced by those processes.
When phospholipids and many other amphipathic lipids are dispersed gently in an aqueous medium they swell, hydrate, and spontaneously form multilamellar concentric bilayer vesicles with layers of aqueous media separating the lipid bilayers. These systems are commonly referred to as multilamellar liposomes or multilamellar vesicles (MLV), and usually have diameters of from 0.2 to 5 μm. Sonication of MLV results in the formation of small unilamellar vesicles (SUV) containing an aqueous solution, bounded by a single lipid bilayer with diameters usually in the range of from 20 to 100 nm. Multivesicular liposomes (MVL) differ from MLV and SUV in the way they are manufactured, in the random, non-concentric arrangement of aqueous-containing chambers within the liposome, and in the inclusion of neutral lipids necessary to form the MVL.
Various types of lipids differing in chain length, saturation, and head group have been used for years in liposomal drug formulations, including the unilamellar, multilamellar, and multivesicular liposomes mentioned above. The neutral lipids used in the manufacture of multivesicular liposomes to date have been primarily limited to triglycerides.
Liposomes in various forms have been prepared by a variety of different processes. However, most such processes are suitable only for laboratory-scale preparation, and are not readily scaled up to batch sizes suitable for commercial production. Thus, a need exists for liposome preparation processes that are suitable for large-scale manufacturing. Among the challenges for the design of an efficient and effective large-scale manufacturing process for (multivesicular) liposomes is the need to bring together unit operations in an efficient manner. Such unit operations include: 1) first emulsification, 2) second emulsification, 3) solvent removal, 4) primary filtration and other ancillary operations necessary for the large-scale production of MVL. The process can also be carried out in an aseptic manner, and such processes are considered desirable.
The rheology of water-in-oil (w/o) emulsions, and specifically the effects of volume fraction and size on viscosity, have been well studied in literature. Multiple emulsion processes are currently used in the pharmaceutical industry to obtain sustained-release drug products. These processes in general begin with the formation of a emulsion with a high volume fraction of dispersed phase (0.6-0.85). High-shear mixing is generally used to obtain a emulsion of high viscosity (from 5-100 times that of the continuous phase) that is stable enough to be further processed.
It is known that the shearing of emulsions in a batch reactor will result in a decrease in the size of droplets in the emulsion. When the shear forces of the mixer exceed the surface tension on discontinuous phase droplets, these forces act to break up the droplets and reduce the mean droplet size. This reduction of droplet size has significant effects on the rheology of the emulsion, with emulsions of smaller droplet size having a higher viscosity. The increase in viscosity with increased mixing time and speed is due to two principle factors. First, smaller droplets have a higher surface tension and are more rigid, resulting in an emulsion with higher viscosity. Second, a decrease in droplet size decreases the mean separation distance between the droplets, resulting in increased hydrodynamic interactions between droplets. The effect of droplet size on emulsion viscosity is magnified as droplet size decreases.
After the w/o emulsion has been formed and it is no longer undergoing any shear forces, many interactions are still occurring within the emulsion. In concentrated dispersions, Brownian motion comes into play, especially if droplet size is small. As Brownian motion causes the droplets to become randomized, collisions between droplets increase and aggregates are formed. In addition, if a w/o emulsion consists of lipids with monolayer membranes of the droplets having their hydrophobic chains facing away from the droplets, attractive hydrophobic interactions occur between droplets, that promote formation of aggregates. When the viscosity of an emulsion containing aggregates is measured at low shear rates, the viscosity is high, while at higher shear rates the aggregates are broken, and the viscosity decreases. Thus, w/o emulsions are shear thinning.
Water in oil in water (w/o/w) emulsions have been prepared by dispersing w/o emulsions into a second aqueous phase. Removal of the solvent of the oil phase by various techniques results in encapsulated materials, present in a second aqueous phase. These materials have found applications in foods, cosmetics, treatment of waste water, and pharmaceuticals.
The removal of solvent from the oil phase to produce such encapsulated materials has been carried out by passing inert gas through the w/o/w emulsion. It has been observed that conventional techniques can result in damage to the encapsulated materials, leading to rupture and loss of material into the second aqueous phase. After the solvent removal step, the encapsulated materials need to be subjected to a primary filtration step.
The primary filtration step has several objectives: exchange the second aqueous solution by a physiologically acceptable solution, concentration adjustment of the multivesicular lipid based particles, and removal of unencapsulated drug. The primary strategy of the prior art for maximizing diafiltration productivity in the manufacture of lipid based particles is to reduce fouling and gel polarization of the membrane. Basic process parameters such as wall shear rate, permeate flux, and transmembrane pressure can be optimized in order to achieve this goal. However, the success of these optimization efforts is limited since the permeate flux drops significantly during the primary filtration process.
In the manufacture of multivesicular liposomes encapsulating various drugs and other active agents, the diafiltration process consumes approximately 60% of the actual process time. Therefore, it is an economical consideration to reduce the time of the diafiltration process to reduce operating cost without compromising the product quality. The approach used in conventional cross-flow systems for reducing the processing time by increasing the wall shear rate or the membrane surface area is counterproductive because multivesicular liposomes are shear sensitive. Increasing the shear rate and membrane surface area, which in turn requires use of a larger pump, results in damaging the particles and reducing the encapsulating yield.
Another concentration adjustment step is often necessary after primary filtration, since the holdup of primary filtration is too great to allow adjustment in one step. Historically, decanting the primary filtration product has been used, although a number of difficulties arise with this approach. Sterility breach is a problem, as well as the necessity of allowing a long resettling time after such a procedure. Concentration adjustment by this method is time-consuming and not particularly accurate.
The products must be sterile for use in human and in many other organisms. Conventional terminal sterilization techniques such as autoclaving and gamma irradiation can damage delicate products, including MVL.
Therefore, due to these problems, new and better methods are required for reducing the process time and shear stress on lipid particles, such as multivesicular particles, during diafiltration. Methods are also required which can reliably produce sterile product without undue product damage.